Lipidomics of the sea sponge Amphimedon queenslandica and implication for biomarker geochemistry.

Demosponges are a rich natural source of unusual lipids, some of which are of interest as geochemical biomarkers. Although demosponges are animals, they often host dense communities of microbial symbionts, and it is therefore unclear which lipids can be synthesized by the animal de novo, and which require input from the microbial community. To address this uncertainty, we analyzed the lipids of Amphimdeon queenslandica, the only demosponge with a published genome. We correlated the genetic and lipid repertoires of A. queenslandica to identify which biomarkers could potentially be synthesized and/or modified by the sponge. The fatty acid profile of A. queenslandica is dominated by an unusual Δ5,9 fatty acid (cis-5,9-hexacosadienoic acid)-similar to what has been found in other members of the Amphimdeon genus-while the sterol profile is dominated by C27 -C29 derivatives of cholesterol. Based on our analysis of the A. queenslandica genome, we predict that this sponge can synthesize sterols de novo, but it lacks critical genes necessary to synthesize basic saturated and unsaturated fatty acids. However, it does appear to have the genes necessary to modify simpler products into a more complex "algal-like" assemblage of unsaturated fatty acids. Ultimately, our results provide additional support for the poriferan affinity of 24-isopropylcholestanes in Neoproterozoic-age rocks (the "sponge biomarker" hypothesis) and suggest that some algal proxies in the geochemical record could also have animal contributions.

NUMTs in the sponge genome reveal conserved transposition mechanisms in metazoans.

The transposition of parts of the mitochondrial (mt) genetic material into the nuclear genome (NUMTs) occurs in a wide range of eukaryotes. Here, we show that NUMTs exist for nearly all regions of the mt genome in the demosponge Amphimedon queenslandica, a representative of the oldest phyletic lineage of animals. Because the sponge NUMTs are small and noncoding, and transposed via a DNA intermediate, as in eumetazoans, we infer that the transpositonal processes underlying NUMT formation in contemporary animals existed in their most recent common ancestor. In contrast to most bilaterians, Amphimedon NUMTs are inserted into regions of high gene density. Given the common features of metazoan NUMTs, the reduction in animal mt genome sizes relative to other eukaryotes may be the product of the mt DNA transposition mechanisms that evolved along the metazoan stem.

The dawn of developmental signaling in the metazoa.

Intercellular signaling underpins metazoan development by mediating the induction, organization, and cooperation of cells, tissues, and organs. Herein, the origins of the four major signaling pathways used during animal development and differentiation-Wnt, Notch, transforming growth factor-beta (TGF-beta), and Hedgehog-are assessed by comparative analysis of genomes from bilaterians, early branching metazoan phyla (poriferans, placozoans, and cnidarians), and the holozoan sister clade to the animal kingdom, the choanoflagellates. On the basis of the incidence and domain architectures of core pathway ligands, receptors, signal transducers, and transcription factors in representative species of these lineages, it appears that the Notch, Wnt, and TGF-beta pathways are metazoan synapomorphies, whereas the Hedgehog pathway arose in the protoeumetazoan lineage, after its divergence from poriferan and placozoan lineages. Examination of the binding domains and motifs present in signaling pathway components of nonbilaterians reveals cases in which signaling interactions are unlikely to be operating in accordance with bilaterian canons. Overall, this study highlights the stability and antiquity of the core cytosolic components of each pathway, juxtaposed with the more variable and recently evolved molecular interactions taking place at the cell surface.

Molecular identification of candidate chemoreceptor genes and signal transduction components in the sensory epithelium of Aplysia.

An ability to sense and respond to environmental cues is essential to the survival of most marine animals. How water-borne chemical cues are detected at the molecular level and processed by molluscs is currently unknown. In this study, we cloned two genes from the marine mollusk Aplysia dactylomela which encode multi-transmembrane proteins. We have performed in situ hybridization that reveals expression and spatial distribution within the long-distance chemosensory organs, the rhinophores. This finding suggests that they could be receptors involved in binding water-borne chemicals and coupling to an intracellular signal pathway. In support of this, we found expression of a phospholipase C and an inositol trisphosphate receptor in the rhinophore sensory epithelia and possibly distributed within outer dendrites of olfactory sensory neurons. In Aplysia, mate attraction and subsequent reproduction is initiated by responding to a cocktail of water-borne protein pheromones released by animal conspecifics. We show that the rhinophore contraction in response to pheromone stimulants is significantly altered following phospholipase C inhibition. Overall, these data provide insight into the molecular components of chemosensory detection in a mollusk. An important next step will be the elucidation of how these coordinate the detection of chemical cues present in the marine environment and activation of sensory neurons.

Convergent antifouling activities of structurally distinct bioactive compounds synthesized within two sympatric Haliclona demosponges.

A wide range of sessile and sedentary marine invertebrates synthesize secondary metabolites that have potential as industrial antifoulants. These antifoulants tend to differ in structure, even between closely related species. Here, we determine if structurally divergent secondary metabolites produced within two sympatric haliclonid demosponges have similar effects on the larvae of a wide range of benthic competitors and potential fouling metazoans (ascidians, molluscs, bryozoans, polychaetes, and sponges). The sponges Haliclona sp. 628 and sp. 1031 synthesize the tetracyclic alkaloid, haliclonacyclamine A (HA), and the long chain alkyl amino alcohol, halaminol A (LA), respectively. Despite structural differences, HA and LA have identical effects on phylogenetically disparate ascidian larvae, inducing rapid larval settlement but preventing subsequent metamorphosis at precisely the same stage. HA and LA also have similar effects on sponge, polychaete, gastropod and bryozoan larvae, inhibiting both settlement and metamorphosis. Despite having identical roles in preventing fouling and colonisation, HA and LA differentially affect the physiology of cultured HeLa human cells, indicating they have different molecular targets. From these data, we infer that the secondary metabolites within marine sponges may emerge by varying evolutionary and biosynthetic trajectories that converge on specific ecological roles.

Mitochondrial diversity of early-branching metazoa is revealed by the complete mt genome of a haplosclerid demosponge.

The first mitochondrial (mt) genomes of demosponges have recently been sequenced and appear to be markedly different from published eumetazoan mt genomes. Here we show that the mt genome of the haplosclerid demosponge Amphimedon queenslandica has features that it shares with both demosponges and eumetazoans. Although the A. queenslandica mt genome has typical demosponge features, including size, long noncoding regions, and bacterialike rRNA genes, it lacks atp9, which is found in the other demosponges sequenced to date. We found strong evidence of a recent transposon-mediated transfer of atp9 to the nuclear genome. In addition, A. queenslandica bears an incomplete tRNA set, unusual amino acid deletion patterns, and a putative control region. Furthermore, the arrangement of mt rRNA genes differs from that of other demosponges. These genes evolve at significantly higher rates than observed in other demosponges, similar to previously observed nuclear rRNA gene rates in other haplosclerid demosponges.

Mox homeobox expression in muscle lineage of the gastropod Haliotis asinina: evidence for a conserved role in bilaterian myogenesis.

Mox homeobox genes are expressed during early vertebrate somitogenesis. Here we describe the expression of Has-Mox, a Mox gene from the gastropod Haliotis asinina. Has-Moxis expressed in the trochophore larva in paraxial mesodermal bands. During larval development, Has-Mox expression remains restricted to mesodermal cells destined to form adult muscle in the foot. This restricted expression of Has-Mox in Haliotis is similar to that observed for vertebrate Mox genes, suggesting a conserved role in myogenesis in deuterostomes and lophotrochozoans. In contrast, Mox is not expressed in muscle lineages in the ecdysozoan representatives Caenorhabditis elegans or Drosophila; the C. elegansgenome has lost Mox altogether. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00427-002-0223-6.

Cytological basis of photoresponsive behavior in a sponge larva.

Ontogenetic changes in the photoresponse of larvae from the demosponge Reneira sp. were studied by analyzing the swimming paths of individual larvae exposed to diffuse white light. Larvae swam upward upon release from the adult, but were negatively phototactic until at least 12 hours after release. The larval photoreceptors are presumed to be a posterior ring of columnar monociliated epithelial cells that possess 120-microm-long cilia and pigment-filled protrusions. A sudden increase in light intensity caused these cilia to become rigidly straight. If the light intensity remained high, the cilia gradually bent over the pigmented vesicles in the adjacent cytoplasm, and thus covered one entire pole of the larva. The response was reversed upon a sudden decrease in light intensity. The ciliated cells were sensitive to changes in light intensity in larvae of all ages. This response is similar to the shadow response in tunicate larvae or the shading of the photoreceptor in Euglena and is postulated to allow the larvae to steer away from brighter light to darker areas, such as under coral rubble-the preferred site of the adult sponge on the reef flat. In the absence of a coordinating system in cellular sponges, the spatial organization and autonomous behavior of the pigmented posterior cells control the rapid responses to light shown by these larvae.

Microsatellite genotyping of individual abalone larvae: parentage assignment in aquaculture.

In aquaculture, microsatellite DNA markers are used to genotype parental broodstock, to assess fertilization success, and to maintain pedigree information for selective breeding. In this study we genotyped individual Haliotis asinina larvae by analyzing a suit of polymorphic microsatellite loci. At least 10 loci can be analyzed from a single abalone veliger larva. We assayed 5 polymorphic loci to identify the parents of individual larvae produced in 3 separate crosses. In all cases, the parents of an individual veliger could be determined from as few as 3 loci. The microsatellite analysis revealed that, in each of our crosses, a single male fathered most of the veligers, despite efforts to normalize the amount of sperm contributed by competing males. These observations suggest that highly controlled breeding practices may be required to ensure that the genetic diversity of an abalone population produced for aquaculture is maintained at the level of diversity of the original broodstock.

Retinoic acid perturbs Otx gene expression in the ascidian pharynx.

In vertebrate embryos, ectopic application of all-trans retinoic acid (RA) alters the expression of Otx genes in the developing midbrain. In conjunction with RA-induced misexpression of other regulatory genes this leads to a loss of anterior CNS. In the ascidian Herdmania curvata, RA primarily inhibits the development of the juvenile pharynx. An ascidian Otx gene, Hec-Otx, is expressed largely in this tissue, associated stomodeal structures and the anterior endostyle of the juvenile. Treatment with 10-6 M RA reduces Hec-Otx mRNA levels in the juvenile to about 12% of normal and is correlated closely with the loss of pharyngeal structures. During embryogenesis the expression of Hec-Otx becomes restricted to cell lineages fated to give rise to the anterior-most nervous system and the stomodeal component of the primordial pharynx. In hatched larvae Hec-Otx transcripts are detected only in the sensory (brain) vesicle. RA reduces Hec-Otx expression in the tailbud stomodeal pharynx primordium/anterior nervous system cell line but not in the larval sensory vesicle, suggesting that RA regulation of Hec-Otx expression is restricted to pharyngeal tissues throughout embryonic and postlarval development. RA does not affect expression of Hec-Pax2/5/8, which is normally expressed within the developing nervous system immediately posterior to Hec-Otx at the tailbud stage, lending support to the proposition that RA does not impact CNS axial patterning. These data combined with those from other chordates suggest that RA regulation of Otx expression in the anterior nerve cord and pharynx is a primitive chordate feature which has been maintained predominantly in pharyngeal tissues in the ascidian.

Neuroectodermal and endodermal expression of the ascidian Cdx gene is separated by metamorphosis.

Ascidians are a group of invertebrate chordates that exhibit a biphasic life history, with chordate-specific structures developing during embryogenesis (dorsal neural tube and notochord) and metamorphosis (pharyngeal gill slits and endostyle). Here we characterize the expression of a caudal/Cdx gene homologue, Hec-Cdx, from the ascidian Herdmania curvata. Vertebrate Cdx genes are expressed at gastrulation and in the posterior of the developing neural tube and endoderm. Hec-Cdx expression is initiated at the earliest stages of gastrulation, with peaks in RNA abundance occurring first during neurulation and tailbud extension and then in 3- to 5-day-old juveniles. Hec-Cdx is expressed in a pair of cells in the anterior lip of the blastopore in the late gastrula which form the most posterior portion of the neural plate. During tailbud formation expression is maintained in and solely restricted to these cells. During metamorphosis expression is localized to the intestine of the juvenile. These data, along with data for the H. curvata Otx gene, suggest that the evolution of the novel ascidian biphasic body plan was not accompanied by a deployment of these genes into new pathways but by a temporal separation of tissue-specific expression.

Expression of a Scr/Hox5 gene in the larval central nervous system of the gastropod Haliotis, a non-segmented spiralian lophotrochozoan.

Hox genes encode a set of evolutionarily conserved transcription factors that regulate anteroposterior patterning mechanisms in insects and vertebrates and are expressed along this axis in a range of bilaterians. Here we present the developmental expression of a Scr/Hox5 gene in the gastropod mollusc Haliotis. In Haliotis, embryogenesis yields a non-feeding trochophore larva that subsequently develops into the veliger larva, which possesses many of the characteristics of the adult body plan. Quantitative RT-PCR analysis reveals that this gene, which is called Hru-Hox5, is first expressed in the trochophore larva. Hru-Hox5 transcript prevalence increases continually through larval development until metamorphic competence develops in the veliger and then again over the first four days of metamorphosis. In situ hybridization reveals that larval expression of Hru-Hox5 is restricted primarily to the primordial and newly formed branchial ganglia, located between the anterior cerebral-pleuropedal ganglionic complex and the posterior visceral ganglia. The expression of Hru-Hox5 in the central region of the abalone CNS is similar to that observed for its orthologue (Lox20) in the leech, suggesting that Hox5 genes were used, along with other Hox genes, to pattern the CNS of the ancestral spiralian lophotrochozoan.

Expression of POU, Sox, and Pax genes in the brain ganglia of the tropical abalone Haliotis asinina.

In gastropod mollusks, neuroendocrine cells in the anterior ganglia have been shown to regulate growth and reproduction. As a first step toward understanding the molecular mechanisms underlying the regulation of these physiological processes in the tropical abalone Haliotis asinina, we have identified sets of POU, Sox, and Pax transcription factor genes that are expressed in these ganglia. Using highly degenerate oligonucleotide primers designed to anneal to conserved codons in each of these gene families, we have amplified by reverse transcriptase polymerase chain reaction 2 POU genes (HasPOU-III and HasPOU-IV), 2 Sox genes (HasSox-B and HasSox-C), and two Pax genes (HasPax-258 and HasPax-6). Analyses with gene-specific primers indicated that the 6 genes are expressed in the cerebral and pleuropedal ganglia of both reproductively active and spent adults, in a number of sensory structures, and in a subset of other adult tissues.

Hemps, a novel EGF-like protein, plays a central role in ascidian metamorphosis.

All chordates share several characteristic features including a dorsal hollow neural tube, a notochord, a pharynx and an endostyle. Unlike other chordate taxa, ascidians have a biphasic life-history with two distinct body plans. During metamorphosis, the larval nerve cord and notochord degenerate and the pharyngeal gill slits and endostyle form. While ascidians, like other marine invertebrates, metamorphose in response to specific environmental cues, it remains unclear how these cues trigger metamorphosis. We have identified a novel gene (Hemps) which encodes a protein with a putative secretion signal sequence and four epidermal growth factor (EGF)-like repeats which is a key regulator of metamorphosis in the ascidian Herdmania curvata. Expression of Hemps increases markedly when the swimming tadpole larva becomes competent to undergo metamorphosis and then during the first 24 hours of metamorphosis. The Hemps protein is localised to the larval papillae and anterior epidermis of the larva in the region known to be required for metamorphosis. When the larva contacts an inductive cue the protein is released, spreading posteriorly and into the tunic as metamorphosis progresses. Metamorphosis is blocked by incubating larvae in anti-Hemps antibodies prior to the addition of the cue. Addition of recombinant Hemps protein to competent larvae induces metamorphosis in a concentration-dependent manner. A subgroup of genes are specifically induced during this process. These results demonstrate that the Hemps protein is a key regulator of ascidian metamorphosis and is distinct from previously described inducers of this process in terrestrial arthropods and aquatic vertebrates.

Hemichordates and deuterostome evolution: robust molecular phylogenetic support for a hemichordate + echinoderm clade.

Hemichordates were traditionally allied to the chordates, but recent molecular analyses have suggested that hemichordates are a sister group to the echinoderms, a relationship that has important consequences for the interpretation of the evolution of deuterostome body plans. However, the molecular phylogenetic analyses to date have not provided robust support for the hemichordate + echinoderm clade. We use a maximum likelihood framework, including the parametric bootstrap, to reanalyze DNA data from complete mitochondrial genomes and nuclear 18S rRNA. This approach provides the first statistically significant support for the hemichordate + echinoderm clade from molecular data. This grouping implies that the ancestral deuterostome had features that included an adult with a pharynx and a dorsal nerve cord and an indirectly developing dipleurula-like larva.

Retinoic acid disrupts anterior ectodermal and endodermal development in ascidian larvae and postlarvae.

In vertebrates, excess all-trans retinoic acid (RA) applied during axis formation leads to the apparent truncation of anterior structures. In this study we sought to determine the type of defects caused by ectopic RA on the development of the ascidian Herdmania curvata. We demonstrate that H. curvata embryos cultured in the presence of RA develop into larvae whose trunks are shortened and superficially resemble those of early metamorphosing postlarvae. Despite RA-treated larvae lacking papillar structures they respond normally to natural cues that induce metamorphosis, indicating that chemosensory functionality previously mapped to the most anterior region of normal larvae is unaffected by RA. Excess RA applied during postlarval development leads to a graded loss of the juvenile pharynx, apparently by respecifying anterior endoderm to a more posterior fate. This structure is considered homologous to the gill slits of amphioxus, which are also lost upon RA treatment. This suggests that RA may have had a role in the development of the pharynx of the ancestral chordate and that this function has been maintained in ascidians and cephalochordates and lost in vertebrates.

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata.

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H. curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae.

We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis. We isolated 12 partial cDNA sequences which were expressed in a stage-specific manner using differential display. We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development. This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)-like repeats and three novel cysteine-rich repeats. Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo. As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis.

A Mox homeobox gene in the gastropod mollusc Haliotis rufescens is differentially expressed during larval morphogenesis and metamorphosis.

We have isolated a homeobox-containing cDNA from the gastropod mollusc Haliotis rufescens that is most similar to members of the Mox homeobox gene class. The derived Haliotis homeodomain sequence is 85% identical to mouse and frog Mox-2 homeodomains and 88.9% identical to the partial cnidarian cnox5-Hm homeodomain. Quantitative reverse transcription-polymerase chain reaction analysis of mRNA accumulation reveals that this gene, called HruMox, is expressed in the larva, but not in the early embryo. Transcripts are most prevalent during larval morphogenesis from trochophore to veliger. There are also transient increases in transcript prevalence 1 and 3 days after the intitiation of metamorphosis from veliger to juvenile. The identification of a molluscan Mox homeobox gene that is more closely related to vertebrate genes than other protostome (e.g. Drosophila) genes suggests the Mox class of homeobox genes may consist of several different families that have been conserved through evolution.